Dehydration/rehydration of derivatized, marker-loaded liposomes on a test device and method of use thereof

ABSTRACT

The present invention relates to a method for making a test device for detecting or quantifying an analyte in a sample. This method involves contacting a membrane with a mixture including derivatized, marker-loaded liposomes, and substantially dehydrating the mixture on the membrane under vacuum pressure at a temperature of from about 4° C. to about 80° C., wherein said mixture further includes one or more sugars in an amount sufficient to promote the stability of the liposomes during dehydration and rehydration. The present invention also relates to a test device and method for detecting or quantifying an analyte in a sample. The test device includes a membrane which includes an immobilized liposome zone, wherein the immobilized liposome zone has bound thereto dehydrated, derivatized, marker-loaded liposomes dehydrated under vacuum pressure at a temperature of from about 4° C. to about 80° C. from a mixture which includes one or more sugars in an amount sufficient to promote the stability of the liposomes during dehydration and rehydration.

This application is a divisional of U.S. patent application Ser. No.10/264,159, filed Oct. 2, 2002, which is a continuation of U.S. patentapplication Ser. No. 09/603,126, filed Jun. 23, 2000, now abandoned,which claims the benefit of U.S. Provisional Patent Application Ser. No.60/140,572, filed Jun. 23, 1999, which is hereby incorporated byreference.

FIELD OF THE INVENTION

The present invention relates to a method of making a test device whichincludes dehydrated, derivatized, marker-loaded liposomes, a test deviceproduced by such a method, and methods of using the test device.

BACKGROUND OF THE INVENTION

Immunoassays, mainly in the form of enzyme-linked immunosorbent assays(ELISAs), have been widely used in the area of clinical diagnosticanalysis (Gosling, Clin. Chem., 36:1408-1427 (1990)). These methodsoffer high specificity, sensitivity, and ease of operation over otherstandard laboratory procedures. However, some of the disadvantages ofthe ELISA format which necessitate further improvement on themethodology include the lengthy time required for antigen-antibodyreaction, reagent additions, enzymatic conversion of substrate, andseveral washing steps between the various operations.

As an alternative to the use of enzymes, liposomes are of interest asdetectable labels in immunoassays because of their potential forimmediate signal amplification. Liposomes are spherical vesicles inwhich an aqueous volume is enclosed by a bilayer membrane composed ofphospholipid molecules (New, Liposomes: A Practical Approach, IRL Press,Oxford (1990)). Previous studies (Plant et al., Anal. Biochem.,176:420-426 (1989); Durst et al., In: GBF Monograph Series, Schmid, Ed.,VCH, Weinheim, F R G, vol. 14, pp. 181-190 (1990) have demonstrated theadvantages of liposome-encapsulated dye over enzymatically producedcolor in the enhancement of signals in competitive immunoassays. Thecapillary migration or lateral flow assay avoids separation and washingsteps and long incubation times and yet attains sensitivity andspecificity comparable to ELISAs. Nevertheless, the methodologies(Siebert et al., Anal. Chim. Acta, 282:297-305 (1993); Roberts et al.,Anal. Chem., 67:482-491 (1995); Siebert et al., Anal. Chim. Acta,311:309-318 (1995); Reeves et al., Trends Anal. Chem., 14:351-355(1995); Rule et al., Clin. Chem., 42:1206-1209 (1996)) involveoperations and solutions that make the handling of the sample andreagents susceptible to errors and more difficult to use for untrainedpersonnel.

The driving force behind the formation of liposomes is hydration, sothat when water is removed from lipid membranes, a shift in the phasetransition occurs and a phase separation of lipids can take placeresulting in aggregation and fusion of the liposomes, thereby losing thebarrier function of the membrane (Lasiv, Biochim. Biophys. Acta,692:501-502 (1982); Mobley et al., J. Control Release, 31:73-87 (1994);Crowe et al., cryobiology, 19:317-328 (1982); Lin et al., Stud.Biophys., 127:99-104 (1988); Crowe et al., J. Bioenerg. Biomembr.,21:77-92 (1989)).

The present invention is directed to overcoming the above-noteddeficiencies in the prior art.

SUMMARY OF THE INVENTION

The present invention relates to a method for making a test device fordetecting or quantifying an analyte in a sample. This method involvescontacting a membrane with a mixture including derivatized,marker-loaded liposomes, and substantially dehydrating the mixture onthe membrane under vacuum pressure at a temperature of from about 4° C.to about 80° C., wherein said mixture further includes one or moresugars in an amount sufficient to promote the stability of the liposomesduring dehydration and rehydration.

The present invention also relates to a test device and its use in amethod for detecting or quantifying an analyte in a sample. The testdevice includes a membrane which includes an immobilized liposome zone,wherein the immobilized liposome zone has bound thereto dehydrated,derivatized, marker-loaded liposomes dehydrated under vacuum pressure ata temperature of from about 4° C. to about 80° C. from a mixture whichincludes one or more sugars in an amount sufficient to promote thestability of the liposomes during dehydration and rehydration. The testdevice preferably includes a capture zone having a first bindingmaterial specific for the analyte bound thereto.

The method for detecting or quantifying an analyte in a test sampleincludes providing a test device in accordance with the presentinvention, wherein the dehydrated, derivatized, marker-loaded liposomesare derivatized with an analyte analog, contacting the test device witha solution of the sample, allowing the solution to migrate through theimmobilized liposome zone and into the capture zone by capillary action,wherein the solution rehydrates the dehydrated, derivatized,marker-loaded liposomes which migrate with the solution into the capturezone by capillary action, permitting any competition to occur betweenany analyte present in the sample and the rehydrated derivatized,marker-loaded liposomes for the first binding material, after saidpermitting, detecting or quantifying the derivatized, marker-loadedliposomes in the capture zone, and correlating the presence or amount ofthe derivatized, marker-loaded liposomes in the capture zone with thepresence or amount of the analyte in the sample.

The method of detecting or quantifying an analyte in a sample alsoincludes providing a test device in accordance with the presentinvention, wherein the dehydrated, derivatized, marker-loaded liposomesare derivatized with a second binding material specific for the analyteand wherein the first binding material binds with a portion of theanalyte other than a portion of the analyte for which the second bindingmaterial is selected, contacting the test device with a solution of saidsample, allowing the solution to migrate through the immobilizedliposome zone and into the capture zone by capillary action, wherein thesolution rehydrates the dehydrated, derivatized, marker-loaded liposomeswhich migrate with the solution into the capture zone by capillaryaction, detecting or quantifying the derivatized, marker-loadedliposomes in the capture zone, and correlating the presence or amount ofthe derivatized, marker-loaded liposomes in the capture zone with thepresence or amount of the analyte in the sample.

In the method of making the test device of the present invention, simpledehydration in a vacuum oven allows the liposomes to rehydrate morequickly because the liposomes remain partially hydrated. In addition,simple dehydration is less expensive, faster, and produces more stableliposomes than freeze-drying procedures.

Moreover, the device can be used directly in the field. The device isused only once, and, therefore, is free from residual environmentalcontaminants other than what may be present in the sample to bemeasured. Samples can be assayed within minutes after collection, withthe results immediately available on-site. In addition, the device andmethod of the invention are less complex than many of the priormaterials and methods. For example, a visible dye can be used as thedetectable marker, eliminating the need for any detection or measurementinstrumentation, and a separate marker or indicator step is not requiredwith any embodiment of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic of a test device of the present invention.

FIG. 2 is a graph showing the recovery of dehydrated liposomes onnitrocellulose strips with varying amounts of external sucrose in theform of a sucrose glaze (▪) and as added excipient in the diluent (●).Nitrocellulose strips incorporating a 4.5 μg antibiotin band were usedwith phosphate buffered saline as the mobile phase. Each point is anaverage of at least three strips and one standard deviation is shown asan error bar.

FIG. 3 is a graph showing the recovery of dehydrated liposomes withvarying internal concentrations of trehalose. Nitrocellulose stripsincorporating a 4.5 μg antibiotin band were used with approximately5.3×10⁸ biotin-tagged liposomes and normal human serum as the mobilephase. For the controls, each point is an average of three strips and,for the dehydrated liposomes, it is an average of four strips and onestandard deviation is shown as an error bar. Internal trehaloseconcentrations: 0 mM (I), 10 mM (II), 50 mM (III), 100 mM (IV), and 200mM (V).

FIG. 4 is a graph showing the recovery after the dehydration/rehydrationprocess for liposomes of varying sizes. Nitrocellulose stripsincorporating a 4.5 μg antibiotin band were used with approximately5.3×10⁸ biotin-tagged liposomes and normal human serum as the mobilephase. Each point is an average of four strips and one standarddeviation is shown as an error bar.

FIG. 5 is a graph showing the relationship between number of liposomesdehydrated on the nitrocellulose strips and gray-scale intensity of theantibody zone. Nitrocellulose strips incorporating a 4.5 μg antibiotinband were used with normal human serum as the mobile phase.

FIG. 6 is a graph showing the effect of gelatin and polyvinylpyrrolidone(PVP) concentrations in blocking reagent on the recovery of dehydratedliposomes and the nitrocellulose strips expressed as signal intensitiesof the antibiotin band produced by intact liposomes. Nitrocellulosestrips incorporating a 4.5 μg antibiotin band were used withapproximately 5.3×10⁸ biotin-tagged liposomes and normal human serum asthe mobile phase. PVP concentrations: 0% (●), 0.05% (▪), 0.2% (♦), 0.5%(▴), and 1% (▾).

FIG. 7 is a dose-response curve for biotin. Nitrocellulose stripsincorporating a 4.5 μg antibiotin band and dehydrated liposomes(approximately 5.3×10⁸ biotin-tagged liposomes/strip) at a point 6 mmbelow the antibiotin band were used with biotin in normal human serum astest sample. Each point is an average of at least three strips, and onestandard deviation is shown as an error bar.

FIG. 8 is a graph showing the stability of liposomes dehydrated onnitrocellulose strips based on the signal intensities obtained over timefor two concentrations of biotin: 10 ng/ml (▪) and 100 ng/ml (●).Nitrocellulose strips incorporating a 4.5 μg antibiotin band anddehydrated liposomes (approximately 5.3×10⁸ biotin-taggedliposomes/strip) at a point 6 mm below the antibiotin band were usedwith biotin in normal human serum as test sample. Each point is anaverage of two strips, and one standard deviation is shown as an errorbar.

DETAILED DESCRIPTION OF THE INVENTION

As described above, the present invention relates to a method for makinga test device for detecting or quantifying an analyte in a sample. Thismethod involves contacting a membrane with a mixture includingderivatized, marker-loaded liposomes, and substantially dehydrating themixture on the membrane under vacuum pressure at a temperature of fromabout 4° C. to about 80° C., wherein said mixture further includes oneor more sugars in an amount sufficient to promote the stability of theliposomes during dehydration and rehydration.

The present invention also relates to a test device for detecting orquantifying an analyte in a sample including a membrane which includesan immobilized liposome zone, wherein the immobilized liposome zone hasbound thereto dehydrated, derivatized, marker-loaded liposomesdehydrated under vacuum pressure at a temperature of from about 4° C. toabout 80° C. from a mixture which includes one or more sugars in anamount sufficient to promote the stability of the liposomes duringdehydration and rehydration. Preferably, the test device also includes acapture zone having a first binding material specific for the analytebound thereto.

The invention further provides a method for detecting or quantifying ananalyte in a test sample by providing a test device in accordance withthe present invention, wherein the dehydrated, derivatized,marker-loaded liposomes are derivatized with an analyte analog,contacting the test device with a solution of the sample, allowing thesolution to migrate through the immobilized liposome zone and into thecapture zone by capillary action, wherein the solution rehydrates thedehydrated, derivatized, marker-loaded liposomes which migrate with thesolution into the capture zone by capillary action, permitting anycompetition to occur between any analyte present in the sample and therehydrated derivatized, marker-loaded liposomes for the first bindingmaterial, after said permitting, detecting or quantifying thederivatized, marker-loaded liposomes in the capture zone, andcorrelating the presence or amount of the derivatized, marker-loadedliposomes in the capture zone with the presence or amount of the analytein the sample.

The method of detecting or quantifying an analyte in a sample alsoincludes providing a test device in accordance with the presentinvention, wherein the dehydrated, derivatized, marker-loaded liposomesare derivatized with a second binding material specific for the analyteand wherein the first binding material binds with a portion of theanalyte other than a portion of the analyte for which the second bindingmaterial is selected, contacting the test device with a solution of saidsample, allowing the solution to migrate through the immobilizedliposome zone and into the capture zone by capillary action, wherein thesolution rehydrates the dehydrated, derivatized, marker-loaded liposomeswhich migrate with the solution into the capture zone by capillaryaction, detecting or quantifying the derivatized, marker-loadedliposomes in the capture zone, and correlating the presence or amount ofthe derivatized, marker-loaded liposomes in the capture zone with thepresence or amount of the analyte in the sample.

By “analyte” is meant the compound or composition to be measured ordetected. A preferred analyte is a nucleic acid molecule.

By “mixture” is meant a solution, suspension, dispersion, or othermixture.

In one embodiment, the method of making the test device of the presentinvention further includes immobilizing a first binding materialspecific for the analyte in a capture zone on the membrane.

By “binding material” is meant a bioreceptor molecule such as animmunoglobulin or derivative or fragment thereof having an area on thesurface or in a cavity which specifically binds to and is therebydefined as complementary with a particular spatial and polarorganization of another molecule—in this case, the analyte.Alternatively, when the target analyte is a nucleic acid molecule, thefirst binding material is a nucleic acid molecule selected to hybridizewith a portion of the target nucleic acid molecule.

Antibody binding materials can be monoclonal or polyclonal and can beprepared by techniques that are well known in the art such asimmunization of a host and collection of sera or hybrid cell linetechnology. The binding material may also be any naturally occurring orsynthetic compound that specifically binds the analyte of interest.

In one embodiment of the present invention, the derivatized,marker-loaded liposomes are derivatized with an analyte analog. Thisembodiment is particularly suitable for use of the test device in acompetitive binding assay. Certain analytes of interest may be sointractable as to make direct conjugation with the liposomeinconvenient, difficult, or even impossible. In such cases, it will benecessary to employ a reactive analog of the analyte of interest toprepare the derivatized liposomes. Thus, by “analyte analog” is meantthe analyte or an analog of which will react with or bind to theliposomes. When an analog is employed, however, it is necessary that theparticular characteristics of the analyte necessary for recognition bythe first binding material in the competition reaction be present in theanalyte analog conjugated with the liposomes.

In another embodiment, the derivatized, marker-loaded liposomes arederivatized with a second binding material. This embodiment isparticularly suitable for use of the test device in a “sandwich” assay.The second binding material may be conjugated to the liposome surface.The second binding material must be bound to the liposomes so as topresent a portion of the second binding material that may be recognizedby the analyte.

Suitable conjugation methods are discussed in U.S. Pat. Nos. 5,789,154,5,756,362, and 5,753,519, which are hereby incorporated by reference.For example, the liposome surface may be activated with thiol groups andcoupled to a maleimide group on the second binding material. Or,conversely, maleimide-activated liposomes and thiol group-activatedsecond binding material may be employed.

When present, the first and second binding materials are selected tobind specifically to separate portions of the analyte. For example, whenthe analyte is a nucleic acid sequence, it is necessary to choose probesfor separate portions of the target nucleic acid sequence. Techniquesfor designing such probes are well-known. Probes suitable for thepractice of the present invention must be complementary to the targetanalyte sequence, i.e., capable of hybridizing to the target, and shouldbe highly specific for the target analyte. The probes are preferablybetween 17 and 25 nucleotides long, to provide the requisite specificitywhile avoiding unduly long hybridization times and minimizing thepotential for formation of secondary structures under the assayconditions. In addition, the first and second binding materials (captureand reporter probes) should not be capable of hybridizing with oneanother. Techniques for identifying probes suitable for the practice ofthe invention are described in Sambrook et al., Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory Press (1989), which ishereby incorporated by reference. A software program known as“Lasergene”, available from DNASTAR, may optionally be used.

In general, to design an assay, the target nucleic acid is extractedfrom a sample, and then amplified by one of a variety of knownamplification techniques. Such amplification techniques includepolymerase chain reaction, ligase chain reaction, and Nucleic AcidSequence Based Amplification (NASBA). See Kievits et al., “NASBAisothermal enzymatic in vitro nucleic acid amplification optimized forthe diagnosis of HIV-1 infection” J. of Virological Methods, 35:273-286(1991), which is hereby incorporated by reference. NASBA, marketed byOrganon-Teknika, a preferred amplification technique when informationregarding the presence or concentration of viable organisms in a sample.

As hereinabove indicated, the methods and test device of the presentinvention include a marker within the interior of the liposomes.Suitable markers include fluorescent dyes, visible dyes, bio- andchemiluminescent materials, enzymatic substrates, radioactive materials,and electrochemical markers. Visible dyes and radioactive materials canbe measured without lysis of the liposomes. Lysis of the liposomes inthe capture zone may be accomplished by applying a liposome lysing agentto the capture zone. Suitable liposome lysing materials includesurfactants such as octylglucopyranoside, sodium dioxycholate, sodiumdodecylsulfate, polyoxyethylenesorbitan monolaurate sold by Sigma underthe trademark Tween-20, and a non-ionic surfactant sold by Sigma underthe trademark Triton X-100, which is t-octylphenoxypolyethoxyethanol.Octylglucopyranoside is a preferred lysing agent for many assays,because it lyses liposomes rapidly and does not appear to interfere withsignal measurement. Alternatively, complement lysis of liposomes may beemployed, or the liposomes can be ruptured with electrical, optical,thermal, or other physical means.

A qualitative or semi-quantitative measurement of the presence or amountof an analyte of interest may be made with the unaided eye when visibledyes are used as the marker. Alternatively, when greater precision isdesired, or when the marker used necessitates instrumental analysis, theintensity of the marker may be measured directly on the membrane using aquantitative instrument such as a reflectometer, fluorimeter,spectrophotometer, etc.

In one embodiment of the invention, a marker which is visible under theassay conditions is used so that the presence and/or amount of analytemay be determined without further treatment and without the use ofinstrumentation, e.g., by use of liposomes containing a dye as themarker.

Alternatively, the methods and test device of the present invention maybe modified to use an electrochemical marker. Suitable electrochemicalmarkers, as well as methods for selecting them and using them aredisclosed in U.S. Pat. No. 5,958,791 to Roberts et al. and co-pendingU.S. patent application Ser. No. 09/315,576, filed May 20, 1999, whichare hereby incorporated by reference.

In a preferred embodiment of the present invention, the membrane is anabsorbent material.

By “absorbent material” is meant a porous material having a pore size offrom 0.05 μm to 50 μm, preferably from 0.45 μm to 5 μm, which issusceptible to traversal by an aqueous medium in response to capillaryforce. Such materials may be natural polymeric materials, particularlycellulosic materials, such as fiber-containing papers, e.g., filterpaper, chromatographic paper, etc.; synthetic or modified naturallyoccurring polymers, such as nitrocellulose, cellulose acetate,poly(vinyl chloride), polyacrylamide, cross linked dextran, agarose,polyacrylate, nylon, activated nylon, polysulfone base modified, etc.;glass fiber, such as borosilicate glass fiber and glass fiber withpolyvinyl alcohol binder (available from Ahlstrom); woven fabric;nonwoven fabric; nonwoven veils; nonwoven materials; polyester fabricsand polyester blend fabrics (available from DuPont, SontaraTechnologies); spunbonded polyester (available from Ahlstrom andDuPont); polypropylene screening fabrics (available from Sefar); rayon;cellulose/rayon; mixed cellulose and glass fiber; polyethersulfone(available from Pall Gelman Sciences); either used by themselves or inconjunction with a support, as described below.

Preferred absorbent materials include nitrocellulose, glass fiber, wovenfabric, nylon, nonwoven material, polyester fabric, rayon, celluloserayon blend, mixed fibers of cellulose and glass, or polyethersulfone.

It is to be understood that the term “nitrocellulose” refers to nitricacid esters of cellulose, which may be nitrocellulose alone, or a mixedester of nitric acid and other acids, and in particular, aliphaticcarboxylic acids having from one to seven carbon atoms, with acetic acidbeing preferred. Such materials, which are formed from celluloseesterified with nitric acid alone, or a mixture of nitric acid andanother acid such as acetic acid, are often referred to asnitrocellulose paper.

The absorbent materials may be polyfunctional or be capable of beingpolyfunctionalized to permit immobilization of the second bindingmaterial.

Materials having a surface area sufficient for supporting the bindingmaterial and any other agents to be immobilized thereon as describedherein may be employed for producing test devices in accordance with thepresent invention.

The test device of the present invention may include one or moreabsorbent materials (i.e., membranes), as described in co-pending U.S.patent application Ser. No. 09/315,576, filed May 20, 1999, which ishereby incorporated by reference. Regardless of the number of absorbentpads or materials employed, it is important that at least that portionof the test strip comprising and between the immobilized liposome zoneand capture zone be made of a non-liposome lysing material. The materialon which the first binding material is immobilized must be capable ofsupporting the immobilization, and the material(s) must allow liquidmigration (lateral flow).

Absorbent materials having high surface areas (such as nitrocellulose)are particularly preferred for some applications in that the firstbinding material and the derivatized, marker-loaded liposomes, may besupported on such materials in high concentrations. It is to beunderstood, however, that the concentration of binding material which isactually used is dependent in part on the binding affinity of the firstbinding material. Accordingly, the scope of the invention is not limitedto a particular concentration of first binding material on the absorbentmaterial.

Application of the first binding material to the membrane may beaccomplished by well-known techniques, for example, by spraying orspotting solutions of this component onto the membrane.

The first binding material can be bound to the membrane by covalentbonding. For example, the material to be bound can be applied directlyto the membrane, and then bonded thereto via ultraviolet radiation.Alternatively, materials can be adsorbed onto the membrane, as long asthe binding of the first binding material to the membrane isnon-diffusive. This will involve contacting the membrane with a solutioncontaining the material to be bound to the membrane and allowing themembrane to dry. In general, this procedure will be useful only wherethe membrane is relatively hydrophobic or has a high surface charge, andsubsequent treatment with proteins, detergents, polysaccharides, orother materials capable of blocking nonspecific binding sites will berequired.

The first binding material is preferably indirectly bound to themembrane in the capture zone of the device. For example, the firstbinding material is preferably labeled with a tag, for example, biotin,and a ligand that specifically binds the tag, for example, streptavidinor anti-biotin antibody, is premixed with the first binding materiallabeled with the tag and then applied to the membrane in the capturezone. Other agents suitable for immobilizing the first binding materialin the capture zone include any compounds or antibodies thatspecifically bind a chosen tag used as a label for the first bindingmaterial, e.g., avidin, anti-fluorescein, anti-digoxin, andanti-dinitrophenyl (DNP).

Before or after application of the first binding material to theappropriate portion on the membrane, the residual nonspecific bindingcapacity of the membrane can be, and preferably is, saturated or blockedwith blocking agents which typically include a combination of threecompounds: proteins, synthetic polymers, and surfactants, and which donot specifically bind the materials to be employed in the assay.Blocking is generally carried out after the first binding material isapplied to the membrane, but it may be possible to block the membranebefore this component is applied depending on the method of application,the particular blocking agent, and membrane employed. Thus, for example,the residual binding capacity of the substrate may be blocked so as toprevent nonspecific binding by the use of bovine serum albumin, asdescribed in Towbin et al., Proc. Nat'l. Acad. Sci., 76:4350 (1979),which is hereby incorporated by reference. The techniques for preventingnon-specific binding are generally known in the art, and such techniquesare also generally applicable to preventing nonspecific binding in theassay of the present invention. Examples of particularly suitabletechniques for blocking with polyvinylpyrrolidone and polyvinylalcoholare described, for example, Bartles et al., Anal. Biochem., 140:784(1984), and in British Patent Specification GB 2204398 A, respectively,which are hereby incorporated by reference. Alternatively, one or moreblocking agents can be incorporated into the buffer solution used towash or carry test components along the membrane(s).

The blocking agents block nonspecific binding sites on the membrane.Thus, preferred blocking agents preferentially bind to the membrane. Theblocking agents are selected from the group consisting of proteinaceousblocking reagents capable of inhibiting binding of molecules having amolecular weight of greater than about 1000 with said membrane andpolymer blocking reagents capable of inhibiting binding of moleculeshaving a molecular weight of less than about 1000 with said membrane.The proteinaceous blocking reagent may be selected from the groupconsisting of gelatin, non-fat dry milk, bovine serum albumin, albuminsfrom other sources, keyhold limpet hemocyanin, casein, gum arabic, fishgelatin, ovalbumin, and horse serum. The polymer blocking reagent may beselected from the group consisting of polyvinylpyrrolidone, polyvinylalcohol, and hydroxypropylmethyl cellulose. The blocking reagent ispreferably a mixture of the above-identified blocking reagents.

Preferably, the blocking agents include a combination ofpolyvinylpyrrolidone and one or a mixture of proteins, such as gelatin,non-fat dry milk, bovine serum albumin, and casein. For liposomesderivatized with small analyte analogs, preferred concentrations ofblocking agents dissolved in, e.g.,Tris(hydroxymethyl)aminomethane-buffered saline, include from about 0.01w/v % to about 1 w/v % polyvinylpyrrolidone and one or a mixture of:from about 0.01 w/v % to about 1 w/v % gelatin, from about 0.01 w/v % toabout 1 w/v % non-fat dry milk, and from about 0.01 w/v % to about 0.05w/v % bovine serum albumin. For liposomes derivatized with largeranalyte analogs, e.g., antibodies and nucleotides, preferredconcentrations of blocking agents dissolved in, e.g.,Tris(hydroxymethyl)aminomethane-buffered saline, include from about0.001 w/v % to about 2 w/v % polyvinylpyrrolidone and one or a mixtureof: from about 0.02 w/v % to about 1 w/v % gelatin, from about 0.02 w/v% to about 1 w/v % non-fat dry milk, from about 0.02 w/v % to about 1w/v % bovine serum albumin, and from about 0.01 w/v % to about 0.3 w/v %casein.

In conjunction with a blocking reagent or reagents, a surfactant may beapplied to the membrane in a concentration sufficient to promotehomogeneous flow of the test solution across the test device, tofacilitate migration of the derivatized, marker-loaded liposomes withoutlysis of the liposomes. Suitable surfactants include Brij™(polyoxyethylene ether), Tween 20™ (polyoxyethylenesorbitanmonolaurate), Triton X-100™ (t-octylphenoxypolyethoxyethanol), sodiumdodecylsulfate, n-octyl--D-glucopyranoside, Span 20™, Nonindet P-40,Chapso™, Turgitol™ and sodium dioxycholate. The concentration of thesurfactant(s) employed in a blocking solution will depend, in part, uponthe liposome composition. In general, surfactants may be incorporated ina concentration of from about 0 to about 0.01 volume percent of theblocking solution, preferably from about 0.001 to about 0.005 volumepercent of the blocking solution. It is important that the concentrationof surfactant applied to the absorbent material be controlled, aspremature lysis of the liposomes may occur if the surfactantconcentration is too high. Preferred surfactants include polyoxyethyleneethers, polyoxyethylenesorbitan mono laurate,t-octylphenoxypolyethoxyethanol, sodium dodecylsulfate,octylglucopyranoside, and sodium dioxycholate.

Blocking agents are applied in a buffer solution to the membrane.Suitable buffers solutions include Tris(hydroxymethyl)aminomethane/HCl(Tris/HCl), Tris/citrate, Tris/maleate, Tris/glycine, phosphate buffer,HEPES, and other biological buffers in the correct pH range.

The membrane can be a single structure such as a sheet cut into strips.The membrane can be mounted on a support material, described more fullybelow. On the other hand, the membrane may provide its own support. Inone embodiment of the invention, the test device includes a strip ofparticulate material bound to a support or solid surface such as found,for example, in thin-layer chromatography. The membrane can be a sheethaving lanes thereon, or be a uniform sheet capable of division intoseparate lanes by physical removal of the absorbent material from thesupport to induce lane formation, wherein a separate assay can beperformed in each lane as shown U.S. Pat. No. 5,958,791 to Roberts etal., which is hereby incorporated by reference. The membranes can be ofa variety of shapes, including rectangular, circular, oval, trigonal, orthe like, provided that there is at least one direction of traversal ofa test mixture by capillary migration. Other directions of traversal mayoccur such as in an oval or circular piece contacted in the center withthe test mixture. However, the main consideration is that there be onedirection of flow from the immobilized liposome zone through the capturezone. In this discussion, strips of membrane support are described byway of illustration and not limitation.

The derivatized, marker-loaded liposomes are introduced onto the testdevice by simple dehydration under vacuum pressure. In particular, amixture comprising derivatized, marker-loaded liposomes and one or moresugars in an amount sufficient to promote the stability of the liposomesduring dehydration and rehydration is applied to an appropriate portionof the membrane. The membrane is then dried in a vacuum oven attemperatures as high as the membrane and liposomes can resist, typicallyat from about 4° C. to about 80° C., more preferably from about 4° C. toabout 50° C., and, most preferably from about 10° C. to about 50° C.,adjusting the drying time accordingly.

As described above, the method of making the test device of the presentinvention includes providing a mixture including derivatized,marker-loaded liposomes and one or more sugars in an amount sufficientto promote the stability of the liposomes during dehydration andrehydration. Suitable sugars include, but are not limited to, sucrose,trehalose, maltose, glucose, lactose, and mixtures thereof. Preferably,the mixture includes from about 0.1M to about 2M sugars, morepreferably, from about 0.7M to about 1M.

In one embodiment, the derivatized, marker-loaded liposomes also includeone or more sugars entrapped within the liposomes, i.e., sugar presentboth inside and outside the vesicles. Suitable sugars are describedabove. Methods for providing liposomes with entrapped sugars and knownin the art and are described in, for example, Example II of the presentapplication, Madden et al., Biochimica et Biophysica Acta, 817:67-74(1985), and Harrigan et al., Chemistry and Physics of Lipids, 52:139-149(1990), which are hereby incorporated by reference.

In a preferred embodiment, the one or more sugars are present solely inthe mixture, i.e., solely on the outside of the vesicles.

As described above, the method of making a test device according to thepresent invention includes substantially dehydrating the mixtureincluding derivatized, marker-loaded liposomes on the membrane undervacuum pressure. Suitable vacuum pressures include from about 5 to about25 psi, more preferably, from about 10 to about 15 psi, with dehydrationtime varying with vacuum pressure.

The migration of the test sample is preferably assisted by introducing awicking reagent, preferably a buffer solution, onto the membrane tocarry the test components along the support. Alternatively, if thesample volume is sufficiently large, it is not necessary to employ aseparate buffer solution.

The support for the membrane where a support is desired or necessarywill normally be hydrophobic, water insoluble, non-porous, and rigid,and usually will be of the same length and width as the absorbent stripbut may be larger or smaller. A wide variety of organic and inorganicmaterials, both natural and synthetic, and combinations thereof, may beemployed, provided only that the support does not interfere with theproduction of signal from the marker. Illustrative polymers includepolyethylene, polypropylene, poly(4-methylbutene), polystyrene,polymethacrylate, poly(ethylene terephthalate), nylon, poly(vinylchloride) poly(vinyl butyrate), glass, ceramics, metals, and the like.

The sizes of the pieces of membrane are dependent on severalconsiderations. The following discussion is primarily focused on stripsof membrane for purpose of illustration and not limitation. As mentionedabove, other shapes such as circular, oval, trigonal, and the like, fallequally within the scope of this invention. The dimensions thereof andother parameters can be determined by those skilled in the art withreference to the disclosure herein.

When capillary flow is predominantly upward, the pore size, length, andthickness of the strip control the amount of mixture that can passthrough the capture zone. If the transfer of a large volume of testmixture is desired, the fluid capacity of the strip beyond the capturezone must be sufficient to accommodate the desired volume.Alternatively, an additional absorbent material, absorbing pad, orsponge, referred to herein as a wicking pad, may be used to contact theend of the membrane beyond the capture zone. A wicking pad may be usedin this manner in situations when it is desirable to pull a largervolume of the test mixture across the test device.

To permit conservation of reagents and provide for samples of limitedsize, the width of the strip will generally be relatively narrow,usually less than 20 mm preferably less than 10 mm. Generally, the widthof the strip will not be less than about 2 mm and will usually rangefrom about 2 mm to 10 mm, preferably from about 3 mm to 6 mm.

As is described in detail below, the test device in accordance with theinvention may be modified for simultaneous multiple analyte detection ordetermination. The length of the strip will depend on the concentrationof the analyte and practical considerations such as ease of handling andthe number of measurement portions on the strip and will be about 4 cmto 20 cm, usually about 5 cm to 15 cm, preferably about 6 to 13 cm butmay be of any practical length. The structure of the strip can be variedwidely and includes fine, medium fine, medium, medium coarse, andcoarse. Selection of the porosity of the material may be based on therate of binding of the components for a given assay.

In one preferred embodiment, a test device according to the presentinvention is made by coating a first binding material onto the membraneat an appropriate portion and then drying the membrane, preferably,under vacuum pressure. Subsequently, the membrane is blocked and dried,preferably under vacuum pressure, for a suitable time determined by themembrane and blocking agents used. The derivatized, marker-loadedliposomes are then applied at an appropriate portion on the membrane andthe membrane is again dried under vacuum pressure.

FIG. 1 shows a test device in accordance with the present invention,depicted immediately after insertion into test sample 208, which is heldin tray 210. As shown in FIG. 1, membrane 212 is mounted on a support214. The test device shown in FIG. 1 includes an immobilized liposomezone 216 which, as described above, has dehydrated, derivatized,marker-loaded liposomes bound thereto and a capture zone 206, which, asdescribed above, has a first binding material for the appropriatederivatized, marker-loaded liposomes bound thereto.

In use in a competition format of the present invention, the membrane212 is inserted into the test sample 208. Wetting of the membrane 212 bycapillary action is allowed to continue at least until capture zone 206is wet (and preferably, until the solvent front reaches the end of themembrane) with test sample 208, respectively. Test sample 208 traversesthe test device into and through immobilized liposome zone 216, wheredehydrated, analyte analog-tagged, marker-loaded liposomes arerehydrated. The test sample 208 continues to traverse the test deviceinto and through capture zone 206, where competition occurs between therehydrated, analyte-analog tagged, marker-loaded liposomes and theanalyte in the test sample for binding sites with the first bindingmaterial bound in the capture zone 206. More liposomes will bind to thecapture zone when there is less analyte in the sample, thus, theintensity of the marker in the capture zone varies inversely with theconcentration of analyte in the test sample.

The test sample may be derived from a wide variety of sources, such asphysiologic fluids, illustrated by saliva, sweat, serum, plasma, urine,tear fluid, spinal fluid, etc., chemical processing streams, food, wastewater, natural waters, soil extracts, etc. Various addenda may be addedto adjust the properties of the test mixture, or of a carrier solutionused as a wicking reagent, depending upon the properties of the othercomponents of the device, as well as on those of the liposomes or theanalyte analog-liposome conjugate, or the analyte itself Examples ofsolution addenda which may be incorporated into test, control, orcarrier solutions or mixtures in accordance with the invention includebuffers, for example, pH and ionic strength, sample or analytesolubilizing agents, such as, for example, nonpolar solvents, and highmolecular weight polymers such as Ficoll®, a nonionic synthetic polymerof sucrose, available from Pharmacia, and dextran.

In the method for detecting or quantifying an analyte in a sample of thepresent invention, the membrane is contacted with test mixture, forexample, by immersing a contact portion of the absorbent material intothe test mixture. Alternatively, the test mixture may be contacted withthe membrane by spotting the test mixture onto the absorbent material ina contact portion, e.g., for lateral flow assays.

The movement of the test components along the membrane(s) is due tocapillary action. This capillary movement along the membrane causes thetest mixture to be carried to and through the capture zone, wheremeasurement of the marker from the liposomes takes place.

In a preferred embodiment, the test device and methods of the presentinvention utilize lateral flow, rather than upward flow, to allow thetest sample and rehydrated, derivatized, marker-loaded liposomes tomigrate the test device.

If quantitative results are desired, wetting of the membrane and anyother absorbent materials, if present, by capillary action is allowed tocontinue until a sufficient volume of test mixture and/or buffersolution has passed through the capture zone to ensure that any analytepresent in the test mixture has reached the capture zone. If detectionalone is desired, less care must be taken to ensure that all analyte hasreached the capture zone. It is possible to “calibrate” run times andbuffer volumes using pre-runs employing calorimetric detection asdescribed herein and in Rule et al., Clin. Chem. 42:1206-1209 (1996),which is hereby incorporated by reference, or electrochemical detectionas described in U.S. Pat. No. 5,958,791 and U.S. patent application Ser.No. 09/315,576, filed May 20, 1999, which are hereby incorporated byreference.

For the most part, relatively short times are involved for the testmixture to traverse the strip. Usually, traversal of the test mixtureover the strip will take at least 30 seconds and not more than 45minutes to 1 hour, more usually from about 1 minute to 10 minutes. Inaccordance with the method of the invention, the signal is rapidly, evenimmediately, detectable.

The conjugate of the second binding material or the analyte-analog andthe marker-encapsulating liposomes may be prepared by proceduresgenerally known in the art, with the particular procedure used in agiven case being dependent upon the liposome components and bindingmaterial employed. Such techniques include covalent coupling,derivatization or activation, and the like. The liposomes may beproduced from a component which has been derivatized with the secondbinding material or analyte analog, whereby the liposomes, whenproduced, are conjugated with the second binding material or analyteanalog. In another procedure, the liposomes, including the marker, maybe initially formed, followed by conjugating the liposomes with thesecond binding material or analyte analog by procedures known in theart.

Liposomes can be prepared from a wide variety of lipids, includingphospholipids, glycolipids, steroids, relatively long chain alkylesters; e.g., alkyl phosphates, fatty acid esters; e.g. lecithin, fattyamines, and the like. A mixture of fatty materials may be employed, suchas a combination of neutral steroid, a charge amphiphile, and aphospholipid. Illustrative examples of phospholipids include lecithin,sphingomyelin, and dipalmitoylphosphatidylcholine, etc. Representativesteroids include cholesterol, chlorestanol, lanosterol, and the like.Representative charge amphiphilic compounds generally contain from 12 to30 carbon atoms. Mono- or dialkyl phosphate esters, or alkylamines; e.g.dicetyl phosphate, stearyl amine, hexadecyl amine, dilaurylphosphate,and the like are representative.

The liposome sacs are prepared in aqueous solution containing the markerwhereby the sacs will include the marker in their interiors. Theliposome sacs may be prepared by vigorous agitation in the solution,followed by removal of the unencapsulated marker. Further details withrespect to the preparation of liposomes are set forth in U.S. Pat. No.4,342,826 and PCT International Publication No. WO 80/01515, both ofwhich are incorporated by reference.

With the test device and method of the invention, one may also assay atest sample for a plurality of analytes such as toxic chemicals orpathogens, or screen for one or more of a plurality of analytes, as isknown in the art.

As a matter of convenience, the present device can be provided in a kitin packaged combination with predetermined amounts of reagents for usein assaying for an analyte or a plurality of analytes. Aside from thetest device with dehydrated, derivatized, marker-loaded liposomes, otheradditives such as ancillary reagents may be included, for example,stabilizers, buffers, and the like. The relative amounts of the variousreagents may be varied widely, to provide for concentration in solutionof the reagents which substantially optimize the sensitivity of theassay. The reagents can be provided as dry powders, usually lyophilized,including excipients, which on dissolution will provide for a reagentsolution having the appropriate concentrations for performing the assay.The kit or package may include other components such as standards of theanalyte or analytes (analyte samples having known concentrations of theanalyte).

The present invention is applicable to procedures and products fordetermining a wide variety of analytes. As representative examples oftypes of analytes, there may be mentioned: environmental and foodcontaminants, including pesticides and toxic industrial chemicals;drugs, including therapeutic drugs and drugs of abuse; hormones,vitamins, proteins, including enzymes, receptors, and antibodies of allclasses; prions; peptides; steroids; bacteria; fungi; viruses;parasites; components or products of bacteria, fungi, viruses, orparasites; aptamers; allergens of all types; products or components ofnormal or malignant cells; etc. As particular examples, there may bementioned T₄; T₃; digoxin; hCG; insulin; theophylline; leutinizinghormones and organisms causing or associated with various diseasestates, such as streptococcus pyrogenes (group A), Herpes Simplex I andII, cytomegalovirus, chlamydiae, etc. The invention may also be used todetermine relative antibody affinities, and for relative nucleic acidhybridization experiments, restriction enzyme assay with nucleic acids,and binding of proteins or other material to nucleic acids.

A device prepared in accordance with the present invention can be usedin a variety of assays, such as competitive binding assays and sandwichassays, as described in U.S. Pat. No. 5,789,154 to Durst et al., U.S.Pat. No. 5,756,362 to Durst et al., U.S. Pat. No. 5,753,519 to Durst etal., U.S. Pat. No. 5,958,791 to Roberts et al., co-pending U.S. patentapplication Ser. No. 09/027,324, filed Feb. 20, 1998, co-pending U.S.patent application Ser. No. 09/034,086, filed Mar. 3, 1998, co-pendingU.S. patent application Ser. No. 09/354,471, filed Jul. 15, 1999, andco-pending U.S. patent application Ser. No. 09/315,576, filed May 20,1999, which are hereby incorporated by reference.

As hereinabove indicated, the assay may be qualitative (presence orabsence of certain level of analyte) or quantitative orsemi-quantitative. The preparation of suitable standards and/or standardcurves is deemed to be within the scope of those skilled in the art fromthe teachings herein.

The methods of the invention, and preparation and use of the test devicein accordance with the invention, are illustrated by the followingExamples.

EXAMPLES Example 1 Materials

Dipalmitoyl phosphatidyl choline (DPPC) and dipalmitoyl phosphatidylglycerol (DPPG) were obtained from Avanti Polar Lipids, Inc. (Alabaster,Ala.). Sulforhodamine B (SRB),N-((6-(biotinoyl)amino)hexanoyl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine, triethylammonium salt (biotinylatedDPPE), and D(+)-biotin were purchased from Molecular Probes (Eugene,Oreg.). Polyvinylpyrrolidone (PVP, 10,000 Da), cholesterol, normal humanserum (NHS), gelatin, bovine serum albumin (BSA), sucrose, trehalose,Tris(hydroxymethyl)aminomethane (Tris), Tween 20, and Sephadex G-50 werepurchased from Sigma Chemical Co., (St. Louis, Mo.). Carnation brandnonfat dry milk (NFDM) was acquired locally. Polycarbonate syringefilters of 3-, 0.4-, and 0.2-μm pore sizes were purchased from OsmonicsLaboratory Products (Livermore, Calif.) and Mylar-supportednitrocellulose membranes with 8-μm pore size were obtained fromSartorius Corp. (Goettingen, Germany) and Millipore Corp. (Bedford,Mass.). The detergent, n-octyl-β-D-glucopyranoside, was obtained fromPfanstiehl Laboratories, Inc. (Waukegan, Ill.).

Example 2 Preparation of Biotin-Tagged, Dye-Loaded Liposomes

Five batches of liposomes, labeled I to V, were prepared to study thestabilizing effect of sugar (trehalose) inside the liposomes. Thebatches were prepared by a modified reverse-phase evaporation method(Siebert et al., Anal. Chim Acta, 282:297-305 (1993); Szoka et al.,Biochim. Biophys. Acta, 601:559-571 (1980); O'Connell et al., Clin.Chem., 31:1424-1426 (1985, which are hereby incorporated by reference)from a mixture of DPPC, cholesterol, DPPG, and biotinylated DPPE in amolar ratio of 4.7:4.8:0.5:0.01. In the lumen of the liposomes wasencapsulated the same concentration of sulforhodamine B (100 mM),increasing amounts of trehalose, and decreasing amounts ofTris/(hydroxymethyl)aminomethane/HCl (Tris/HCl) buffer, pH 7.0, tomaintain an osmolality value of 827 mOsmol/kg for each preparation (seeTable 1).

TABLE 1 Biotin-Tagged Liposome Characteristics Liposome preparation I IIIII IV V VI VII VIII Mean diameter* (nm) 321 353 294 375 336 242 300 384Liposome concentration 5.5 × 10¹² 3.6 × 10¹² 7.9 × 10¹² 6.3 × 10¹² 3.1 ×10¹² 3.1 × 10¹² 1.5 × 10¹² 8.3 × 10¹¹ per ml Trehalose concentration 010 50 100 200 0 0 0 (mM) SRB concentration 100 100 100 100 100 175 175175 (mM) Biotin (molecules per 719 870 603 982 788 409 628 1029liposome) Osmolality 827 827 827 827 827 204 204 204 (mOsmol/kg)*Measured value.Briefly, the procedure involved dissolving the lipids in a solventmixture containing chloroform:isopropyl ether:methanol (6:6:1, v/v). Thetemperature was kept at 45° C. throughout the entire process. Theencapsulant was added and the mixture was sonicated for 5 minutes undera low flow of nitrogen. The organic phase was removed under vacuum on arotary evaporator. Another aliquot of encapsulant solution was added,and the liposome preparation was sonicated for an additional 30 minutes.The liposomes were then extruded sequentially three times through 3-,0.4-, and 0.2-μm polycarbonate syringe filters arranged in tandem. Toremove the unencapsulated dye, the preparation was gel filtered on aSephadex G-50-150 column (Sigma Chemical Co., (St. Louis, Mo.))equilibrated with the appropriate buffer which similar in osmolality tothe encapsulant solution and dialyzed against 1 liter of the samebuffer.

Three other batches of liposomes, labeled VI to VIII, were prepared tostudy the effect of liposome size on stability. These batches wereprepared by the same reverse-phase evaporation method using anencapsulant containing 175 mM sulforhodamine B in 0.02 M Tris/HCl, pH7.5, buffer (osmolality of encapsulant equals 204 mOsmol/kg). Thedifferences in the procedure for the three batches were in the length ofsonication time to which each batch was subjected and the pore size ofthe filters used which resulted in differences in the liposome sizes.Longer sonication times produced smaller liposomes.

All liposomes were stored in buffers that were iso-osmolar or up to 100mOsmol/kg above their respective encapsulant at 4° C. in the dark untilneeded.

Example 3 Characterization of the Liposomes

The mean diameter of the liposomes was measured by a laser-basedlight-scattering particle-size analyzer (PCS sizing system from MalvernInstruments Ltd, Malvern, UK). Fluorescence intensity measurements werecarried out on a McPherson Model SF 750 spectrofluorometer (Acton,Mass.) operated at excitation and emission wave-lengths of 543 and 596nm, respectively.

Example 4 Preparation of Antibiotin-Coated Test Strips

Antibiotin antibodies were immobilized on sheets of nitrocellulosemembranes (45×160 mm) in a 2.5-mm-wide band, 15 mm from the bottom ofthe sheet using a microprocessor controlled Linomat IV TLC SampleApplicator (Camag Scientific, Inc., Wrightsville Beach, N.C.). Theantibody-coated membranes were dried in a vacuum oven (15 psi) at roomtemperature for 1 hour and then immersed in various blocking agentsdissolved in Tris-buffered saline (TBS; 0.02 M Tris/HCl, 0.15 M NaCl,and 0.01% NaN₃, pH 7.0) for 1 hour. The blocked membranes were dried atroom temperature in a vacuum oven for different time periods and cutinto strips (5×45 mm). Each test strip contained 4.5 μg of antibiotin inthe capture zone. The strips were stored at 4° C. in vacuum-sealedplastic bags until ready for use.

Glazed strips were prepared by applying sucrose solutions of differentconcentrations, using the Linomat IV sample applicator, onto an area 6mm below the antibiotin band on nitrocellulose membrane sheetspreviously coated with antibiotin antibodies and blocked with 0.2% PVPand 0.01% gelatin in TBS. The glazed membranes were air-dried for 1 hourbefore being cut into 5×45-mm strips and stored in vacuum-sealed plasticbags until ready for use.

Example 5 Dehydration of Liposomes on Antibiotin-Coated Strips

Liposome solutions were prepared using diluents containing differentexcipients in phosphate-buffered saline (PBS; 0.01 M KH₂PO₄/K₂HPO₄, 0.15M NaCl, and 0.01% NaN₃, pH 7.0). One microliter of the diluted liposomesolution was applied at a point 6 mm below the antibiotin band andimmediately covered with aluminum foil to prevent lysis ofsulforhodamine B (SRB)-loaded liposomes by light (SRB-encapsulatedliposomes were observed to be slightly sensitive to light). The stripswere dried in a vacuum oven (15 psi) at room temperature for 2 hours.

Strips without liposomes were also dried in the vacuum ovensimultaneously with the test strips for use as controls. The purpose wasto obtain the same degree of hydration in both the test strips and thecontrols.

For the experiments where the effects of sugar glaze under thedehydrated liposomes were investigated, 1 μL of the liposome solutionwas applied on top of the sugar glaze and also dried in the vacuum ovenat room temperature for 2 hours.

Example 6 Recovery Test Protocol

The recovery test was performed by inserting the test strips into a testtube, or a holder device, containing PBS (pH 7.4) or NHS as carrier ormobile phase. When the solution front reached the top of the strip, thestrip was removed and air-dried. The antibiotin capture zone collectedthe biotin-tagged liposomes that remained intact after thedehydration/rehydration process. The control consisted of applying 1 μLof the same liposome solution onto the control strips at a point 6 mmbelow the antibiotin band and, without drying, immediately insertingthem into the test tubes containing the mobile phase.

Example 7 Biotin Assay Protocol

The assay was performed by inserting the test strips, which containedthe dehydrated biotin-tagged, SRB-loaded liposomes at a zone below theantibiotin band, into test tubes holding the liquid sample or thecalibration samples of biotin in PBS, TBS, or NHS. When the solutionfront reached the top of the strip, the strip was removed and air-dried.

Example 8 Detection and Quantitation

The color intensity of the antibiotin capture zone was measuredoptically. For a more accurate quantitation of the red coloration of theband, a Hewlett-Packard ScanJet IIc desktop scanner (Palo Alto, Calif.)and scan analysis densitometry software (Biosoft, Ferguson, Mo.)installed on a Macintosh Performa 636CD were used. This approach allowedthe conversion of the red coloration into a gray-scale reading thatcould be quantified (Siebert et al., Anal. Chim. Acta, 282:297-305(1993), which is hereby incorporated by reference). The results arerepresented in arbitrary units of the gray-scale intensity or as apercentage of recovery of the dehydrated/rehydrated liposomes whencompared with the control, that is, assigning a value of 100% to thecontrol.

Example 9 Liposome Characteristics

Sulforhodamine B fluorescence is self-quenching when encapsulated athigh concentration, thus the integrity of the liposomes can bedetermined by measuring the fluorescence of a liposome solution beforeand after lysis of the liposomes. Total lysis of the liposomes wasobtained by the addition of a solution of n-octyl β-D-glucopyranoside toa final concentration of 30 mM. The stability over time can be studiedby measuring the percentage of free dye in the preparation. Themeasurement of dye encapsulated in the liposomes and the determinationof liposomes size by the laser scattering method allowed the calculationof the liposome characteristics (see Table 1). All calculations wereperformed as previously described (Siebert et al., Anal. Chim. Acta,282:297-305 (1993), which is hereby incorporated by reference), takinginto account that the DPPE-biotin is 0.1 mol % of the total lipids andassuming that the dye encapsulated was equal in concentration to theoriginal dye solution used, that the bilayer thickness is 4 nm, and thatthe average surface head-group area of DPPC molecules and cholesterolmolecules in a mixed bilayer is 0.71 and 0.19 nm², respectively(Israelachvili et al., Biochim. Biophys. Acta, 389:13-19 (1975), whichis hereby incorporated by reference).

Example 10 Recovery of Dehydrated/Rehydrated Liposomes on NitrocelluloseStrips

Basically, two major points were important in the process ofdehydration/rehydration of liposomes on nitrocellulose strips. The firstwas the determination of the appropriate blocking agents and dryingconditions for the membranes, and the second was the study of thecomponents of the liposome preparation, which included the encapsulant(internal), the excipient in the diluent (external), and thecharacteristics of the liposomes.

Initial testing involved the use of varying concentrations of differentblocking agents prepared in Tris-buffered saline. Polyvinylpyrrolidone(PVP), gelatin, bovine serum albumin (BSA), and non-fat dry milk (NFDM)were tested at 0.02, 0.1, and 0.5%, and Tween 20 at 0.002 and 0.02%.Liposome batch VII (no internal trehalose) was diluted with phosphatebuffered saline (PBS) (no excipients) and 1 μL was dehydrated on theblocked nitrocellulose strips below the antibiotin band as describedabove. Among the blocking agents tested, PVP provided the bestprotection of the liposomes after the dehydration/rehydration cycle, asindicated by the highest signal obtained at the antibiotin capture zone.Listed in order of decreasing effectiveness in preserving the dehydratedliposomes are the blocking agents at their individual optimalconcentrations and the respective percentage of recovery of theliposomes obtained: 0.5% PVP (14.9% recovery)>0.1% gelatin (8.7%recovery)>0.02% BSA (3.7% recovery)>0.1% NFDM (2.5% recovery). The useof unblocked membranes (Tris-buffered saline-treated) and those blockedwith Tween 20 resulted in complete lysis of the liposomes when the driedliposomes were rehydrated by the mobile phase.

Drying the blocked membrane overnight (14 hours) in the vacuum oven (15psi) at room temperature resulted in very dry membranes. During themigration of the mobile phase, it was observed that the liquid took along time (ca. 15 minutes) to migrate past the capture zone. Inaddition, the mobile phase migrated preferentially along the strip edgeswhich reduced the chances of liposomes binding in the capture zone. Bydecreasing the drying time of the blocked membranes to 8 hours, thecarrier solution migrated more rapidly and uniformly through the capturezone and the signal on the antibiotin band was improved.

Example 11 Effect of Sucrose Glaze on the Recovery of the Liposomes

Liposome batch I (no internal trehalose) was used to study the effect ofsucrose glaze on the recovery of liposomes. The concentration of sucrosetested ranged from zero to 4.4 μmol/strip. The results showed that thesucrose glaze, placed on the strips prior to the application of theliposome preparation for dehydration, provided a dramatic improvement inthe recovery of the dehydrated/rehydrated liposomes. The glaze greatlydiminished the aggregation observed previously at the point ofapplication of the liposomes after the mobile phase passed by, and itconsiderably increased the intensity at the antibiotin band whichindicated higher recovery of intact liposomes. The optimal amount ofsucrose glaze was observed to be ca. 1.5 μmol/strip (see FIG. 2) andresulted in 70% recovery. At sucrose glazes greater than ca. 2.2μmol/strip, the aggregation at the point of application of the liposomescompletely disappeared, but the signal intensity at the antibiotin banddecreased.

Example 12 Components of the Liposome Preparation

1. Sugar outside of the liposomes. Due to the observation that the sugarglaze greatly improved the preservation of the dehydrated/rehydratedliposomes, the effect of sugar added as excipient in the diluent for theliposome preparation prior to its application onto the strips wasevaluated and compared with the results of the sugar glaze.Concentrations of sucrose and trehalose ranging from 0.07 to 0.6 M wereprepared in PBS, pH 7.4, and used as diluent for liposome batch I (nointernal trehalose). Without external sugar, aggregation appeared at thepoint of application of the liposomes after the mobile phase migratedpast the dehydrated liposomes, and the antibiotin zone bound only veryfew liposomes, i.e., many of the liposomes lysed upon rehydration. Asthe concentration of sugars was increased, the aggregation at the pointof application diminished until aggregation completely disappeared andconcomitantly, the recovery of the dehydrated liposomes increased.Sucrose was observed to have a slightly better preservation effect thantrehalose. An added advantage of choosing sucrose over trehalose is thelower cost. A study with higher concentrations of sucrose was carriedout and the optimal concentration of external sucrose needed forstabilizing the dehydrated liposomes was observed to be in the range of0.7 to 1.0 M or 0.7 to 10 μmol/μL/strip (see FIG. 2). At sucroseconcentrations higher than 1.0 M, the recovery of the dehydratedliposomes decreased. In addition, the flow rate of the mobile phase withthe high sucrose content became very slow after contacting thedehydrated liposomes. The uniformity of the capture zone also decreased.The slower flow rate can be explained by the increase in viscosity ofthe mobile phase due to the sucrose.

The results shown in FIG. 2 indicated that less sucrose (0.7 to 1.0μmol/μL/strip) was needed in the diluent for the liposomes compared tothe sucrose glaze (1.5 μmol/strip) to achieve protection of thedehydrated liposomes. Moreover, with sucrose in the diluent, a higherdegree of recovery was obtained. Considering these results and the easeof adding sucrose to the diluent versus application of the sugar glaze,the former method was chosen for the subsequent work.

2. Sugar inside of the liposomes. According to the literature (Crowe etal., Arch. Biochem. Biophys., 242:240-247 (1985); Madden et al.,Biochim. Biophys. Acta, 817:67-74 (1985); Harrigan et al., Chem. Phys.Lipids, 52:139-149 (1990); Crowe et al., In: Liposome Technology,Gregoriandis, Ed., CRC Press, Boca Raton, Fla., vol. 1, pp. 229-270(1993), which are hereby incorporated by reference), trehalose must bepresent both inside and outside of the vesicles to stabilize theliposomes during lyophilization and dehydration processes. For thepurpose of finding the optimum internal concentration of trehalose, fivebatches of liposomes were prepared entrapping the followingconcentrations of trehalose: 0, 10, 50, 100, and 200 mM (see Table 1).The different batches of liposomes were diluted with PBS containing 0.42M sucrose before application onto the nitrocellulose strips fordehydration. The results shown in FIG. 3 indicated that, in the presenceof sucrose outside the liposomes, trehalose encapsulated in theliposomes did not improve the stability of the liposomes undergoing thedehydration/rehydration process on nitrocellulose membranes. Internaltrehalose concentrations higher than 100 mM may even have adestabilizing effect on the liposomes. These liposome stability resultsobtained on nitrocellulose membranes are in contrast to the positiveeffect of both internal and external trehalose reported for lyophilizedliposomes (Crowe et al., Arch. Biochem. Biophys., 242:240-247 (1985);Madden et al., Biochim. Biophys. Acta, 817:67-74 (1985); Crowe et al.,In: Liposome Technology, Gregoriandis, Ed., CRC Press, Boca Raton, Fla.,vol. 1, pp. 229-270 (1993), which are hereby incorporated by reference)and dehydrated liposomes (Harrigan et al., Chem. Phys. Lipids,52:139-149 (1990), which is hereby incorporated by reference). Onepossible explanation might be differences in the lipid composition ofthe liposomes used in this study versus the ones reported. Another mightbe the different physical characteristics of these liposomes whendehydrated on membranes compared to those in the frozen or dehydratedstate.

Example 13 Effect of Liposome Size on Stability

Three preparations of liposomes which were 242, 300, and 384 nm indiameter (batches VI, VII, and VIII in Table 1) were studied. Althoughseveral reports (Harrigan et al., Chem. Phys. Lipids, 52:139-149 (1990);Crowe et al., In: Liposome Technology, Gregoriandis, Ed., CRC Press,Boca Raton, Fla., vol. 1, pp. 229-270 (1993); Crowe et al., Biochim.Biophys. Acta, 939:327-334 (1988), which are hereby incorporated byreference) showed that vesicles larger than 200 nm in diameter were lessstable than smaller ones, the stability of the larger liposomes wasinvestigated because of the previous observation that greater signalintensities could be obtained with larger liposomes. While the resultsin FIG. 4 show that there is a slight increase in the recovery for thesmaller liposomes, liposomes with diameters ranging from 242 to 384 nmmay be recovered successfully after a dehydration/rehydration process,making them useful in a one-step strip assay.

Example 14 Effect of Number of Liposomes

The results of FIG. 5 show a positive linear correlation betweengray-scale intensity of the binding zone and the number of liposomesdehydrated on the nitrocellulose strip. However, the percentage ofliposomes recovered upon rehydration remained constant. That is,liposomes within the range needed to produce a distinguishable signal(2.5×10⁸ to 2×10⁹ liposomes/strip) provided the same percentage ofrecovery (75%) after the dehydration/rehydration process. Although ahigher signal was obtained as the number of dehydrated liposomesincreased, greater amounts of analyte were needed to compete with theanalyte tags on the liposomes. Thus, to keep the limit of detection low,one must select an amount of liposomes that will give a strong enoughsignal for the zero standard and yet be able to provide a signal for thelowest possible concentration of analyte that is distinguishable fromthe zero standard. The number of liposomes that provided the bestresponse curve was between 7×10⁸ and 9×10⁸ liposomes per stripcontaining 4.5 μg of antibiotin at the binding zone.

Example 15 Effect of Osmolality Inside of the Liposomes

The comparisons among liposomes batches I to V (all with the sameinternal osmolality of 827 mOsmol/kg) and batches VI to VIII (all withinternal osmolality of 204 mOsmol/kg) in FIGS. 3 and 4 showed similarrecoveries after the dehydration/rehydration process. This observationmakes possible the application of this approach to a wide range ofliposomes encapsulating different markers, since every water-solublecompound to be encapsulated as a marker will have its own osmolalityvalue. This would also be of potential importance for development of amulti-analyte assay.

Example 16 Optimization of Components for Maximal Recovery of DehydratedLiposomes

The observation that external sugar was necessary for the preservationof the dehydrated liposomes necessitated a redetermination of theoptimal concentrations of the blocking agents needed for maximalrecovery of the dehydrated sucrose-enriched liposomes. Experiments usingPVP and gelatin by themselves or mixed in different proportions wereperformed using liposome batch I (no internal trehalose) enriched with0.44 M sucrose in the diluent (0.44 M was used before 0.7 to 1.0 M wasdetermined to be the optimal range). FIG. 6 shows the effects ofcombinations of PVP and gelatin on the recovery of the dehydratedliposomes. Gelatin by itself is able to preserve a large number ofliposomes, but the uniformity over the capture zone is poor. The resultsindicate that the membranes blocked with 0.2% PVP and 0.01% gelatinprovided the highest recovery of the dehydrated/rehydrated liposomes.Later experiments showed no significant difference between 0.1 and 0.2%PVP when used with 0.01% gelatin. PVP was observed to be essential inproviding the best flow rate and homogeneity over the antibiotin band.

Based on the foregoing observations, for best results, liposomes withdiameters smaller than 384 nm and without internal sugars were made upin diluent containing between 0.7 and 1 M sucrose prior to applicationonto an area below the antibody band on the nitrocellulose stripsblocked with 0.1 or 0.2% PVP and 0.01% gelatin in TBS.

Example 17 Test Performance

Table 2 shows the reproducibility of the test with strips that containeddehydrated liposomes together with control strips (liposomes freshlyapplied) using PBS or normal human serum (NHS) as the carrier.

TABLE 2 Test Performance Mobile phase PBS NHS Strips ControlDe/rehydrated Control De/rehydrated Mean recovery (%) 100 71.9 100 79.6Standard deviation 6.0 5.0 7.2 6.8 (%) Coefficient of 6.0 7.0 7.2 8.6Variation (%) N 21 26 26 25The percentage of recovery for strips with dehydrated liposomes is theaverage of the intensities of the antibiotin band of all the test stripsin comparison to the intensity of the antibiotin band of the controls.Using NHS as the carrier, the recovery was significantly higher thanwith PBS. From these results, a range of 70 to 80% recovery wasestablished with an acceptable coefficient of variation (CV) of ≦8%. Theanalysis time, which consisted of the time from the contact of the teststrip containing the dehydrated liposomes with the sample solution untilthe solution reached the end of the strip, was less than 6 minutes.Additional time for the calorimetric quantitation with the scanner, thetechnique used in the laboratory, was needed. For a simpler and moreuser-friendly approach, a meter to measure the color intensity at aspecific wavelength based on reflectometry could be used. Most simply, avisual estimation of the color intensity, using appropriate calibrantsor a color intensity chart, would obviate the need for anyinstrumentation.

Example 18 Biotin Strip Immunoassay

The test was based on the principle of a competitive immunoassay. As theliquid sample migrated up the test strip, it rehydrated the driedbiotin-tagged, SRB-loaded liposomes and continued to migrate toward theimmobilized antibody zone, where competition occurred between thebiotin-conjugated liposomes and the analyte in the sample for bindingsites on the antibody. More liposomes will bind to the antibiotin zonewhen there is less biotin in the sample, thus, the intensity of thecolor on the antibody capture zone varies inversely as the concentrationof the biotin in the sample. A dose-response curve for biotin in normalhuman serum in shown FIG. 7. This dose-response curve exhibits thesigmoidal shape of competitive immunoassays. From this curve, the limitof detection was determined to be 6 ng/mL (24.6 nM) of biotin withgreater than 95% confidence. While this concentration is greater thanthe clinically relevant level in blood/serum, it is expected thatfurther improvements will lower the detection limit to the requisitelevel.

To determine if different amounts of binding protein (antibody) on thestrip would affect the limit of detection and the working range of theassay, strips were coated with 1, 2.5, and 4.5 μg of antibiotin. Thestrips with only 1 μg of antibiotin did not have enough antibody to bindliposomes to give a visually discernible band. The response curvesobtained using strips containing 2.5 and 4.5 μg of antibiotin showed thesame detection limit, but a slight improvement in the working range wasobserved with the higher concentration of antibiotin on the strip.

Example 19 Stability over Time of Test Strips Containing DehydratedLiposomes

The strips with dehydrated liposomes were stored in vacuum-sealedplastic bag at 4° C. and protected from light. A calibration curve usingstrips that had been kept at 4° C. and protected from light for a yearshowed identical response to that obtained when the strips were freshlyprepared. The results obtained with two biotin standards, 10 and 100μg/mL biotin, tested over a period of 1.2 years using the stored teststrips, are shown in FIG. 8. The insignificant change in the responsesobtained for the standards indicated that the dehydrated liposomes onthe NC strips were stable for at least one year. For the 10 and 100 μg/Lbiotin standards, the % CV values obtained are 5.50 (n=8) and 5.14(n=7), respectively.

Thus, the feasibility of developing a rapid one-step strip immunoassayusing dehydrated liposomes was demonstrated. This technique provides asimple and convenient format for point-of-care and field assay devices.The dehydration of dye-loaded liposomes in the presence of a highconcentration of external sugar on a nitrocellulose membrane blockedwith PVP and gelatin provided 70-80% recovery of the liposomes uponrehydration by the sample solution. The optimal external sucroseconcentration was determined to be 0.7-1.0 M. Relatively largevariability in liposome size, internal osmolality, encapsulantconcentration, and liposome number did not affect the recovery of theliposomes, which greatly simplified the production process for theassay. The blocking conditions disclosed herein are the optimum achievedfor biotin as the analyte, but modifications would be necessary forother analytes, especially proteins.

Although the invention has been described in detail for the purpose ofillustration, it is understood that such detail is solely for thatpurpose, and variations can be made therein by those skilled in the artwithout departing from the spirit and scope of the invention which isdefined by the following claims.

1. A test device for detecting or quantifying an analyte in a test sample, the test device comprising a membrane, the membrane comprising: a contact portion at or proximate to a first end of the membrane; an immobilized liposome zone positioned adjacent the contact portion, wherein the membrane at the immobilized liposome zone has a sugar glaze on its outer surface and the immobilized liposome zone has bound thereto dehydrated, derivatized, marker-loaded liposomes dehydrated under vacuum pressure at a temperature of from about 4° C. to about 80° C.; an electrochemical measurement portion at a location on the membrane which is positioned away from the first end, wherein the electrochemical measurement portion comprises an electrochemical detector cell comprising a first conductor having a plurality of fingers, and a second conductor having a plurality of fingers, wherein the fingers of the first conductor are interdigitated with the fingers of the second conductor and are positioned on a glass substrate removably positioned adjacent and in contact with a surface of the membrane at the electrochemical measurement portion, and wherein the conductors induce redox cycling; and a liposome lysing portion segregated from the contact portion and immobilized liposome zone and having a liposome lysing agent bound thereto, wherein the liposome lysing portion either is positioned between the contact portion and the electrochemical measurement portion, or partially or completely coincides with the electrochemical measurement portion.
 2. The test device according to claim 1, wherein the glass substrate comprises silicate glass.
 3. The test device according to claim 1, wherein the membrane further comprises a competitive binding portion positioned between and segregated from the contact portion and the liposome lysing portion and having a binding material for the analyte bound to the competitive binding portion.
 4. The test device according to claim 3, further comprising a reference electrode disposed on the membrane between the competitive binding portion and the electrochemical measurement portion.
 5. The test device according to claim 4, wherein the reference electrode is electrically connected to the first conductor and the second conductor.
 6. The test device according to claim 1, wherein the membrane further comprises a capture portion positioned between and segregated from the contact portion and the liposome lysing portion and having a capture probe selected to at least partially hybridize with a portion of the analyte bound to the capture portion.
 7. The test device according to claim 1, wherein the first conductor and the second conductor are electrically connected to one another.
 8. The test device according to claim 1, further comprising a support material on which membrane is mounted.
 9. The test device according to claim 1, further comprising a reference electrode disposed on the membrane between the contact portion and the electrochemical measurement portion.
 10. The test device according to claim 9, wherein the reference electrode is electrically connected to the first conductor and the second conductor.
 11. The test device according to claim 1, wherein either or both of the first conductor and the second conductor comprise one or more materials selected from the group consisting of platinum, gold, graphite, silver, and titanium.
 12. The test device according to claim 1, wherein each of the first conductor and the second conductor comprise from 2 to 1000 fingers.
 13. The test device according to claim 1, wherein the fingers of the first and second conductors are each from about 1 μm to about 20 μm wide and are spaced from about 0.5 μm to about 10 μm apart. 